植物生理

Argonaute (AGO) proteins recruit small RNAs to form effector complexes of RNA interference (RNAi), collectively termed RNA-induced silencing complexes (RISCs). Here, we describe detailed protocols for the purification of AGO complexes and their associated small RNAs, using Arab ...

Plant microRNA (miRNA) processing requires at least two cleavage steps of respective precursors. The first cleavage step is from pri-miRNA to pre-miRNA, and the second cleavage step is from pre-miRNA to mature miRNA. Using northern blot analysis, we previously showed that the RNase III enzyme ...

MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at the posttranscriptional level. Moreover, recent studies reported several miRNAs asso ...

The methods described herein first highlight the strategies that were used to discover a biotic stress-associated miRNA. This involved (1) the selection of transcripts that were more abundant in transgenic plants expressing viral-derived suppressors of RNA silencing and transcr ...

Short, interfering RNAs (siRNAs) arise from the processing of long double-stranded RNA (dsRNA) by Dicer enzymes. Dicers generate siRNA duplexes by successive hydrolysis of both strands of the dsRNA phosphodiester backbone at positions determined by measuring 21–24 nucleotides fr ...

The characterization of gene function typically includes a detailed analysis of loss-of-function alleles. In model plants, such as Arabidopsis thaliana and rice, sequence-indexed insertion collections provide a large resource of potential null alleles that can often be easily ac ...

Next-generation sequencing technologies have a substantial impact on a broad range of biological applications. Like many other groups, we use these new technologies, especially SBS (Sequence-By-Synthesis), for deep profiling of small RNA molecules in plants. Small RNAs are 21–24 nuc ...

A spatial and temporal analysis of miRNA accumulation by in situ analyses is the prerequisite of understanding the precise biological functions of miRNAs. Since miRNAs are very short molecules, their in situ analysis is technically demanding. Here, we describe a protocol for miRNA in situ de ...

This chapter describes a stepwise protocol for Agrobacterium-mediated sorghum genetic transformation. Immature embryos from sorghum plants were used as the target explants. The Agrobacterium strain LBA4404, carrying a “super-binary” vector, was used in this protocol. Agroba ...

This chapter describes a method for rapid gene expression assays in Arabidopsis. Three distinct plasmids are codelivered into leaves by microparticle bombarment. The first one carries a transacting factor gene driven by the cauliflower mosaic virus (CaMV) 35S promoter. The second pla ...

Large DNA molecules (l00 kb) are extremely sensitive to mechanical shearing m aqueous solution. Therefore, classical DNA extractron procedures from living tissues generally do not allow recovery of DNA fragments larger than 50‐100 kb m size. In recent years, technical improvements have ...

Abstract The importance of determining spatial distribution and expression of biomolecules in living organisms has been increasingly recognized because of the relevance to biological function. High resolution spatial information at the level of the cell or organism can be obtai ...

The science of plant breeding has been a major force in the development of new and improved cultivars and hybrids of major agricultural crops. All people have benefited from these developments. In conventional plant breeding, one must usually find the trait of interest within the same or closely ...

Gene overexpression as a means to determine plant gene function has been used almost since the first plant transformation protocols became viable. The goal of these experiments, as in classical genetic experiments, is to observe any phenotypic change associated with changing the expre ...

As aerobic chemoorganotrophs, most Agrobacterium strains will grow on a wide range of complex and defined media. Methods commonly used for the culture and storage of other chemoorganotrophs will usually work for agrobacteria as well. Problems with culture or strain maintenance will oc ...

A binary vector is a standard tool in the transformation of higher plants mediated by Agrobacterium tumefaciens. It is composed of the borders of T-DNA, multiple cloning sites, replication functions for Escherichia coli and A. tumefaciens, selectable marker genes, reporter genes, and oth ...

The genetic manipulation of Agrobacterium tumefaciens is used to facilitate studies of bacterial gene functions or as a first step in introducing genetic material into transformable plant cells through the use of T-DNA binary vectors. Three methods are commonly used. Transformation ...

Agrobacterium tumefaciens has been widely used to transform numerous plant species. Frequently, investigators want to place more than one gene into Agrobacterium in order to manipulate various bacterial functions during plant genetic transformation. These genes are frequen ...

The ability of Agrobacterium to transform plants and other organisms is under highly regulated genetic control. Two Virulence (Vir) proteins, VirA and VirG, function as a two-component regulatory system to sense particular phenolic compounds synthesized by wounded plant tissues. I ...

Agrobacterium is routinely used as a tool for moving genetic constructs into plant cells. The successful use of Agrobacterium as a tool for the genetic engineering of plant cells often requires the manipulation and analysis of nucleic acids present in recombinant Agrobacterium strains. ...